LEGO-Lipophosphonoxins: A Novel Approach in Designing Membrane Targeting Antimicrobials

The alarming rise of bacterial antibiotic resistance requires the development of new compounds. Such compounds, lipophosphonoxins (LPPOs), were previously reported to be active against numerous bacterial species, but serum albumins abolished their activity. Here we describe the synthesis and evaluation of novel antibacterial compounds termed LEGO-LPPOs, loosely based on LPPOs, consisting of a central linker module with two attached connector modules on either side. The connector modules are then decorated with polar and hydrophobic modules. We performed an extensive structure–activity relationship study by varying the length of the linker and hydrophobic modules. The best compounds were active against both Gram-negative and Gram-positive species including multiresistant strains and persisters. LEGO-LPPOs act by first depleting the membrane potential and then creating pores in the cytoplasmic membrane. Importantly, their efficacy is not affected by the presence of serum albumins. Low cytotoxicity and low propensity for resistance development demonstrate their potential for therapeutic use.


■ INTRODUCTION
Most of the antibiotics in use today are derivatives of natural products of actinomycetes and fungi. 1 Medicinal chemistry has played a key role in modifying natural products to optimize their pharmacological properties while minimizing toxicity. 2 Nevertheless, bacterial pathogens resistant to currently available drugs already cause at least 700,000 deaths globally a year, including 230,000 deaths from multidrug-resistant tuberculosis, a figure that could increase to 10 million deaths globally per year by 2050 under the most alarming scenario if no action is taken. 3 Many current antibiotics were developed during the golden era of antibiotic drug discovery (1940s−1980s), and most target five biosynthetic processes that occur in actively growing bacteria: the biosynthesis of proteins, RNA, DNA, peptidoglycan, and folic acid. Most of these classical antimicrobial strategies are not effective for eradicating persistent infections in which bacteria are quiescent, and strains resistant to these antibiotics readily emerge. 4 An attractive target for the development of antibacterial compounds is the cytoplasmic membrane as the composition of bacterial and mammalian cell membranes differs, resulting in different biophysical properties. 5 In contrast to majority of classical antibiotics requiring metabolically active bacterial cells, membrane targeting antimicrobials are capable of also killing persistent (dormant) bacteria. A number of membrane-active compounds are already known.
Antimicrobial peptides (AMPs) and host defense peptides (HDPs) are examples of membrane-active compounds with enhanced affinity for the negatively charged prokaryotic membranes with strong electrical potential gradients as prerequisites for cellular entry or direct disruption of the bacterial cell membrane. 6,7 These peptides are the first line of defense in many multicellular organisms and possess a broad range of biological activities, including antibacterial, antifungal, antiviral, anticancer, antiplasmodial, antiprotistal, insecticidal, spermicidal, and immunomodulatory activities. As AMPs target the cell membrane of the microorganisms for direct antimicrobial action, bacteria find it difficult to develop resistance.
Despite so many advantages, peptide antibiotics have had relatively little clinical success largely due to their in vivo toxicity, limited bioavailability, and large production costs. The only peptide antibiotics that are being used clinically are shown in Figure 1 and include polymyxin B (a lipopeptide obtained from Bacillus polymyxa), colistin (polymyxin E, also from B. polymyxa), gramicidin (a linear polypeptide derived from Bacillus brevis), and daptomycin (a cyclic anionic lipopeptide produced by Streptomyces roseosporus). 5 To overcome the limitations of AMPs, many researchers have turned their focus to peptidomimetics (small molecule membrane targeting agents [SMMTAs]) that reproduce the critical biophysical characteristics of AMPs, such as positive charge, hydrophobicity, and amphipathicity, while being relatively simple to synthesize and exhibiting better pharmacokinetic properties. Examples of SMMTAs are (i) LTX-109 ( Figure 1), a first-in-class chemically synthesized, peptidemimetic drug that is stable against protease degradation and represents a new approach to the serious challenge of Staphylococcus aureus nasal decolonization; 8,9 (ii) ceragenin CSA-13 that is active against a broad spectrum of Gram-negative and Gram-positive bacteria; 10,11 (iii) synthetic retinoid CD437 that exhibits potent in vitro bactericidal activity against S. aureus strains including the MRSA strain MW2 but not against Gramnegative species; 12 (iv) XF-73 (exeporfinium chloride), which is a novel antistaphylococcal membrane-active photosensitizing porphyrin derivative that is active against a broad spectrum of Gram-positive bacteria; 13,14 and (v) brilacidin (PMX-30063) that belongs to arylamide foldamers and has shown therapeutic benefits in clinical trials. 15,16 Finally, lipophosphonoxins (LPPOs) are promising antibacterial compounds that belong among SMMTAs and that we developed several years ago. LPPOs are small amphiphilic molecules bearing positive charge(s). Their general structure ( Figure 2) consists of four modules: (i) a nucleoside module (NM), (ii) a polar module (PM), (iii) a hydrophobic module (HM), and (iv) a phosphonate connector module (CM) that holds together modules i−iii. This first-generation LPPO (LPPO I) 17,18 demonstrated excellent bactericidal activity against various Gram-positive species, including multiresistant strains such as vancomycin-resistant enterococci or methicillinresistant S. aureus. The minimum inhibitory concentration (MIC) values were in the 1−12 mg/L range, while their cytotoxic concentrations against human cell lines were above this range (IC 50 60−100 mg/L). We have shown that at their bactericidal concentrations, LPPOs act via the disruption of the cytoplasmic membrane. 17 However, LPPO I compounds are ineffective against Gramnegative bacteria. By redesigning the iminosugar module so that it bears more positive charges, we developed the second generation of LPPOs (LPPO II) with increased efficacy (MIC <1−6 mg/L) against Gram-positive species and an extended antibacterial activity range that now also includes serious Gramnegative pathogens such as clinically relevant strains of Escherichia coli, Pseudomonas aeruginosa, and Salmonella Enteritidis. 19 LPPO II cause serious damage to the bacterial cell membrane, efflux of the bacterial cytosol, and cell disintegration. 20 Employing model membranes (liposomes and black lipid membranes), we demonstrated that LPPO II act by creating pores in the membrane. Furthermore, LPPO II were shown to  be well tolerated by live mice when administered orally (2000 mg/kg) and to cause no skin irritation in rabbits.
Importantly, using several of the most potent LPPO I and LPPO II (Figure 2), we failed to select Bacillus subtilis, Enterococcus faecalis, or Streptococcus agalactiae strains resistant against compound 1 (LPPO I) and, in addition, a P. aeruginosa strain resistant to LPPO II compound 2, while strains resistant to known conventional antibiotics (rifampicin and ciprofloxacin, respectively) readily emerged in control experiments. Recently, LPPO II were evaluated as additives in polymethylmethacrylate (PMMA) bone cements, preventing infections, 21 and as an antibacterial component of a polycaprolactone electrospun nanofiber dressing capable of reducing S. aureus induced wound infection in mice. 22 Nevertheless, despite all the beneficial properties of LPPOs, their antibacterial activity is abolished in the presence of serum albumins. To address this limitation, we performed structure− activity relationship (SAR) studies. First, we designed compounds lacking the nucleoside module (LPPO III). The antibacterial activities of these compounds, however, were also inhibited by serum albumins. Subsequent SAR studies inspired by symmetrical peptidomimetics 15,23−30 resulted in new modular structures that were loosely based on LPPOs. The pivotal part of these structures was the linker module (LM) connecting the two parts of the molecule. Hence, these compounds were termed linker-evolved-group-optimized-LPPOs (LEGO-LPPOs). LEGO-LPPOs can be synthesized in a few easy steps. The best-performing LEGO-LPPOs displayed equal or better antimicrobial properties and significantly better selectivity than known LPPOs. Importantly, the antibacterial activity of LEGO-LPPOs was not affected by serum albumins. LEGO-LPPOs acted by depolarizing the microbial membrane and displayed pore forming activities. Similar to LPPO I and II, resistance to LEGO-LPPOs was not detected. Finally, additional tests demonstrated their safety and potential as therapeutics. ■ RESULTS LPPO III. As the first step in the optimization process of LPPOs, we removed the nucleoside module (5′-uridyl moiety) from LPPO II and created a set of asymmetrical compounds, LPPO III.
In LPPO III, NM was replaced with various simple ester groups to obtain a series of new derivatives 10a−o (Table 1) employing the same chemistry (Scheme 1) as in our original study. 19 Diethyl (4) or dimethyl vinylphosphonate (6) served as starting material. R 1 and R 2 groups were subsequently installed by reaction of monoethyl (5) or monomethyl vinylphosphonate with the appropriate hydroxyl derivative R 1 OH and R 2 OH either using TPSCl (2,4,6-triisopropylbenzenesulfonylchloride) as condensing agent or via phosphonochloridate generated from monomethyl vinylphosphonate with oxalylchloride/DMF. Next, protected PM was introduced by Michael addition to the vinylphosphonate 8 double bonds. Finally, protecting groups were removed from PM by treatment with 0.5 M methanolic HCl to afford final LPPO 10a−o.
Next, tests of biological activities of these compounds were performed. Some of the derivatives (e.g., 10a−c) retained their antibacterial activity (Table 2). However, their hemolytic activities were also relatively high, decreasing the selectivity of these compounds. Unfortunately, as in the case of original LPPO I and LPPO II, the antibacterial activities of LPPO III were lost in the presence of bovine serum albumins (BSA).

LEGO-LPPO.
The failure of LPPO III prompted us to redesign the skeleton of LPPOs. We were inspired by dimeric, often symmetrical peptidomimetics. Examples of these peptidomimetics are fatty acids comprising lysine conjugates, 23 phenylalanine conjugated lipophilic norspermidine derivatives, 24 antimicrobial arylamide oligomers 25,26 including brilacidin, 15 and others. 27−30 This resulted in symmetrical LEGO-LPPO structures, which were based on a new modular system depicted in Figure 3. We varied mostly HM and/or LM. As PM, bis(3-aminopropyl)amino was used in the majority of the compounds, and ethylphosphonate was exclusively used as CM.
The synthesis of LEGO-LPPO is depicted in Scheme 2. Dimethyl vinylphosphonate (6) served again as the starting material. First, HM (R) was attached by the reaction of monomethyl vinylphosphonate with the appropriate hydroxyl derivative ROH either using TPSCl as condensing agent or via phosphonochloridate generated from monomethyl vinylphosphonate with oxalylchloride/DMF. Tetrabutylammonium salt of monoester 12 obtained by aqueous pyridine promoted demethylation of 11 reacted with α,ω-dibromoalkane, introducing LM (X). Next, protected PM (Y) was introduced by Michael addition to the vinylphosphonate 13 double bonds. Finally, protecting groups from PM were removed by treatment with 0.5 M methanolic HCl to yield final LEGO-LPPO 14−83 (Scheme 2 and Table 3). For all compounds, cLogD values at pH 7.4 were calculated, and the gradient chromatography hydrophobicity index (CHIg) of the compounds was measured by the linear gradient HPLC method and calculated based on the retention time and acetonitrile composition as described previously. 31 Antibacterial Activities of LEGO-LPPO. MIC Values and Hemolytic Activity. All LEGO-LPPO compounds were tested against a panel of Gram-positive and -negative species and evaluated for cytotoxicity by determining their hemolytic activity against erythrocytes (HC 50 ) ( Table 4 and Table S4 for MBC values). The best compounds displayed excellent MICs ranging from <1 to 8 mg/L, while their HC 50 were at least 20− 100 times above their respective MIC values. Importantly, the presence of serum albumin only slightly, if at all, affected their antibacterial activity. To extend the testing to real clinical  Figure 4. Briefly, LEGO-LPPOs were typically able to reduce viable cell counts to zero within several hours. The effect was concentration dependent. In the assay, compound 38 showed kinetics comparable to those of control antibiotics, daptomycin, and colistin, respectively.
Persister Killing Assay CCCP. Next, we characterized the ability of the compound with the most pronounced antibacterial activity, 38, and three different comparable antibiotics (colistin, daptomycin, and cell wall-acting ampicillin/sulbactam [AMS]) to kill persister cells. Three bacterial species were used (E. coli, P. aeruginosa, and S. aureus). Concentrations of the tested substances corresponded to 1×, 5×, and 10× MIC. Figure 5 shows that 38 was able to kill persister cells, superior to AMS in all cases, equal to colistin in the case of E. coli, and less efficient than colistin with P. aeruginosa and daptomycin with S. aureus. Still, the detected activity is of note and reveals a potential for the use of 38 also against persister cells.
Resistance to LEGO-LPPO Is Difficult to Emerge. To evaluate the potential of LEGO-LPPOs for long-term use, we used two of the already characterized LEGO-LPPO, 25 and 38, and attempted to select bacterial strains resistant to these compounds using the clinically relevant pathogen P. aeruginosa ( Figure 6). With LEGO-LPPO, we were unable to select resistant strains. On the contrary, with control antibiotics, a 4fold increase in MIC was observed by the end of the selection experiment for ciprofloxacin (from 0.5 to 2 mg/L), and a 32-fold increase of MIC was seen for ceftazidime (from 4 to 128 mg/L).
Effect of LEGO-LPPO on Cell Integrity. As LEGO-LPPOs are derived from LPPOs that function by compromising the cell envelope, we next addressed the effect of LEGO-LPPOs on cell integrity. As the first approach, we used scanning electron microscopy (SEM) to assess the cell envelope damage, testing the effect of compounds 25 and 38 on S. aureus. Compared to untreated controls, visible damage of the cells induced by the presence of the compounds was detected (Figure 7), including  loss of integrity and cytoplasmic content (empty vessels) and the presence of tubular structures, possibly membranous nanotubes that extrude from dying/dead cells. 33 This result was consistent with LEGO-LPPOs targeting the cytoplasmic membrane. Membrane Potential. Next, to characterize the mechanistic action of LEGO-LPPOs, we selected three compounds, each representing a different class of LEGO-LPPO with respect to activity/selectivity: (i) compound 33 with activity only against Gram-positive bacteria and excellent selectivity, (ii) compound 38 with a broad spectrum of antibacterial activity and good selectivity, and (iii) compound 71 with a broad spectrum of antibacterial activity and poor selectivity (strong hemolytic activity).
We started by looking at the effect of LEGO-LPPOs on the membrane potential−electrical potential gradient that bacteria maintain across the plasma membrane. We rationalized that if LEGO-LPPOs act on the cell membrane, their effect could be first detected as loss of the membrane potential. Both 38 and 71 were able to rapidly depolarize the plasmatic membrane of both S. aureus and E. coli cells within a few seconds after the addition at a concentration of 2.5 mg/L ( Figure 8). In contrast, 33 was almost ineffective in promoting changes in bacterial membrane potential within the time course of the experiment. This is consistent with its relatively high MIC values compared to 38 and 71.
We note that the loss of the membrane potential did not cause immediate cells death as Figure 4 shows no decrease in cell count within the first few hours of the experiment. This is consistent with the notion that cells generate the largest membrane potential when metabolically active 34 and suggests that LEGO-LPPO first cause metabolic arrest followed by a slower killing effect.
Membrane Permeabilization: PI Assay. Next, we tested the ability of the same LEGO-LPPOs as used in the previous experiment (33, 38, 71) to permeabilize E. coli membranes to allow passage of the large molecules of propidium iodide (PI). Both 38 and 71 promoted a relatively slow PI influx ( Figure 9) in a concentration-dependent manner. Surprisingly, at the LEGO-LPPO concentration, which had been effective in membrane depolarization (2.5 mg/L, Figure 8), the propidium cation did not pass through the E. coli membranes. When higher concentrations were used, we observed a slow influx of PI into the bacterial cells. 33 was unable to permeabilize the E. coli plasmatic membrane for PI even at the highest concentration used (10 mg/mL). This was not surprising as the MIC value of 33 against E. coli is 32 mg/L. The dramatic differences between 38 and 71 vs 33 then may be due to variability in the affinity of these compounds for target membranes.
From the functional perspective, the permeabilizing effect requires higher concentrations or longer times than 80 min used in this experiment. This is consistent with the time interval in time-kill assays ( Figure 4) where it took ∼12 h for 38 (at 2 mg/ L) to reduce the bacterial population by 6 orders of magnitude.

LEGO-LPPOs Make Pores in the Planar Phospholipid
Bilayer. As both 38 and 71 showed the ability to depolarize bacterial membranes (i.e., promote the fluxes of small inorganic ions) but a relatively slow membrane permeabilization for propidium iodide, we characterized the pore-forming activity of 38 on planar phospholipid bilayers (made of negatively charged 1,2-diphytanoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DPhPG)) that are more susceptible to permeabilizing effects than natural membranes ( Figure 10). In the presence of 38 at 2.5 mg/L, we observed pores of two different types: highly fluctuating unstable pores ( Figure 10B, upper line) or regular pores ( Figure 10B, lower line) of distinct conductance about 10 pS in 1 M KCl. At lower concentrations of 38, we observed mostly regular pores (not shown). The most frequent conductance of ∼10 pS ( Figure 10A) suggested the presence of relatively narrow and well-defined membrane pores. This was consistent with the slower efflux rate of the cellular contents than in the case of more aggressive pore-forming compounds.
Interaction of LEGO-LPPO with a Model Membrane: Leakage from Phospholipid Vesicles. Next, we tested the relationship between the LEGO-LPPO activity and specific membrane composition. We used phospholipid vesicles created by binary mixtures without solvents using dioleoylphosphatidylethanolamine (DOPE), dioleoylphosphatidylglycerol (DOPG), and dioleoylphosphatidylcholine (DOPC). We selected the combinations of DOPE/DOPG (2:1) and DOPC/DOPE (2:1) mimicking the composition of plasmatic membrane of Gram-negative bacteria and mammalian cells, respectively. The unilamellar vesicles loaded with carboxyfluorescein (CF) revealed membrane permeabilization by increases in CF fluorescence after its leakage. In DOPE/DOPG vesicles, we observed a higher membrane disrupting activity of 38 and 71 in comparison to 33 (Figure 11), which corresponds with the ability of these molecules to permeabilize the E. coli plasmatic membrane (cf. Figure 8 and Figure 9). The leakage kinetics of 38 displayed a monophasic behavior ( Figure 11A), whereas 71 showed typical sigmoidal kinetics ( Figure 11B), suggesting differences in the mechanisms of membrane permeabilization by these two molecules. When tested on DOPC/DOPE (2:1) vesicles, 71 showed dramatically enhanced membrane disruptive activity ( Figure 11B) in terms of the initial leakage rate and final maximum leakage, which explains its high hemolytic activity (lysis of red blood cells containing phosphatidylcholine in their membrane).
Tests of Cytotoxicity of LEGO-LPPO on Mammalian Cell Cultures, and Skin and Eye Irritation Tests. To conclude the characterization of LEGO-LPPOs, we performed a final block of experiments, further addressing the level of their cytotoxicity and safety as potential therapeutics, using several approaches. For the first approach, we selected a large set of LEGO-LPPOs to detect their potential for cytotoxicity toward HepG2 cells. These cells are nontumorigenic, display high proliferation rates, and are routinely used in drug metabolism and hepatotoxicity studies. 35,36 The results are summarized in Table 6; the cytotoxic Scheme 2. Synthesis of LEGO-LPPO Compounds a a concentrations of the compounds were significantly above the respective MIC values.
Next, as LEGO-LPPOs have a high potential also as topical drugs, we performed in vitro skin and eye irritation tests using two compounds with excellent antibacterial activities and low hemolytic, cytotoxic activities, 25 and 38. The compounds were tested at concentrations of 20 and 200 mg/L in both tests. For potential skin irritability, we used the reconstructed human epidermis model test (RhEM). 37 For potential eye irritability, we used the reconstructed human cornea-like epithelium (RhCE) 38 test method designed to identify chemicals not requiring classification and labeling for eye irritation or serious eye damage. In both tests, LEGO-LPPOs performed excellently, showing no detrimental effects (Tables S1 and S2). According to the RhEM test, both tested LEGO-LPPOs could be considered to be nonirritant to skin in accordance with the UN GHS ″no category″. The tissue viabilities after 1 h exposure and 42 h posttreatment incubation were determined as higher than 60% compared to the negative control, and identical ″no category″ results were also obtained for LEGO-LPPOs in the RhCE test.
Next, we assessed the in vivo effect of compounds 25 and 38 at concentrations of 100 and 200 mg/L in the standard skin irritation test (according to OECD 404 and EN ISO 10993-1:2003) and at a concentration of 100 mg/L in the standard eye irritation test (according to OECD 405) performed with rabbits. For both 25 and 38, the skin or eyes of all animals were without any adverse effects (for details see the Experimental Section and SI).
Finally, the most promising compound 25 was used in tests of the maximum tolerated dose (MTD) in mice, probing its compatibility with systemic use. The compound was administered orally (p.o.) and subcutaneously (s.c.). MTD for p.o. administration was >200 mg/kg of body weight and >15 mg/kg of body weight for s.c. administration. During the observation period after both p.o. and s.c. administration, no clinical signs were observed in the animals, and during the necropsy, no gross pathological changes were detected.

■ DISCUSSION
In this study, we present the design, synthesis, and characterization of novel antibacterial compounds termed LEGO-LPPOs. The modular structure of LEGO-LPPO allows easy, inexpensive synthesis in a few steps, an important property for prospective therapeutics. 39 LEGO-LPPOs are active against both Grampositive and -negative bacteria with cytotoxicity levels significantly above their MIC values. Furthermore, LEGO-LPPOs are also active against multiresistant strains of S. aureus as well as clinical isolates, and resistance to these compounds was not detected in the P. aeruginosa model. Importantly, antibacterial activities of LEGO-LPPOs are virtually unaffected by serum albumins, unlike their predecessors, LPPO II. 19 Finally, in vitro and in vivo skin and eye irritability tests with selected LEGO-LPPOs proved their safety for potential topical applications. Furthermore, oral and subcutaneous administration of 25 demonstrated its potential for systemic use.
Based on mechanistic studies, the action of LEGO-LPPOs can be likened to the hunting strategy of spiders: trap first, kill later. LPPO-LEGO first act by depleting the membrane potential that is important for generating energy, active metabolism, and cell division. 40 This is the fast step, the trapping. It is followed by a slower step, the killing, where LPPO-LEGOs form pores in the membrane, compromising its integrity. Both the membranepotential-depleting and pore-forming activities of the tested compounds correlated with respective MIC values. The pore-  forming activity makes LPPO-LEGOs members of SMMTAs. 5,9,11,41,42 The activity and selectivity of LPPO-LEGOs depend on their structure. While polar modules A, B, and C appear to have equivalent effects, it is the hydrophobicity index of the compounds that is most correlated with their activity and selectivity. The least hydrophobic compounds 14   As HM, we have evaluated saturated and unsaturated linear alkyl chains, aromatic phenylethyl groups, and cyclic moieties including cyclohexyl and adamantyl. The aromatic phenethyl HM appeared to have too low hydrophobicity, so only compound 72 with a 10 carbon atom long LM exhibited some antibacterial activity (and only against S. aureus and S. epidermidis), albeit with very low hemolytic activity (HC 50 > 500 mg/L). Cyclohexylpropyl HM used in combination with a 6 carbon atom long LM in compound 40 exhibited very good antibacterial activity (MIC 0.5−4 mg/L against most of the bacterial strains except for E. faecium where MIC = 16 mg/L) with low hemolytic activity (HC 50 = 183 mg/L). Among the compounds with linear alkyl chains as LM, compound 25 was the best performer with MIC 1−4 mg/L against most of the bacterial strains except for E. faecium where MIC = 64 mg/L and with very low hemolytic activity (HC 50 = 264 mg/L). Promising antibacterial activity (broad spectrum) and good selectivity was exhibited by compounds 23, 31, 32, and 49 containing the adamantyl moiety in their LM. Finally, LEGO-LPPOs with low MIC and high hemolytic values generally displayed cLogD between 1 and 2, suggesting good solubility and permeability, 43 similarly to a number of newly approved drugs. 44 To summarize, we discovered a new scaffold based on original LPPOs upon which we synthesized compounds with significantly better selectivity than second-generation LPPOs (where best compounds exhibited HC 50 values in the range of 16−30 mg/L).
To conclude, LEGO-LPPOs are a new class of compounds with broad-spectrum antibacterial activities suitable for further development into potential therapeutics. We tested in detail several selected LEGO-LPPOs, of which the two most promising compounds, 25 and 38, were tested most thoroughly. Of these two compounds, 25 showed a higher therapeutic potential due to its low cytotoxicity. However, other LEGO-LPPO compounds also displayed favorable properties (e.g., 23, 31, and 32) and will be included in future evaluations as experiments on animal models are already being designed. These most promising compounds will undergo a more detailed SAR study focused mostly on the connector module to obtain fully stereochemically defined or completely achiral species.   TFA in 50% aq. methanol, C = methanol) or without buffer. All final compounds were lyophilized from water. The purity of the final compounds was greater than 95%. Purity of final compounds was determined via LC−MS analysis using an Acquity UPLC coupled with a Xevo G2 XS QTof (Waters) and column XBridge 50 × 2.1 mm, 1.7 μm (Waters). Mass spectra were recorded on an LTQ Orbitrap XL (Thermo Fisher Scientific) using ESI ionization. NMR spectra were   5)) fluorescence. The increase in intensity represents the depolarization of the bacterial membrane. Both 38 and 71 were able to depolarize rapidly the plasmatic membrane of both Gram-positive and Gram-negative bacteria within a minute after their addition. In contrast, 33 was virtually ineffective in promoting changes in bacterial membrane potential. General Methods. General Method A1: Removal of the Phosphonate Methyl Ester Group. Methyl vinylphosphonate (1 mmol) was dissolved in 60% aqueous pyridine (20 mL), and the reaction mixture was stirred at 60°C for 24 h. The reaction mixture was concentrated in vacuo at a temperature below 40°C, and the residue was dissolved in ethanol (20 mL) and passed through a column of Dowex 50 H + form (5 g). The column was washed with EtOH (40 mL). The solvent was removed in vacuo. The product was obtained by column chromatography on silica gel using a linear gradient of solvent system H1 (ethyl acetate/acetone/ ethanol/water 4:1:1:1) in ethyl acetate.
General Method A2: Removal of the Phosphonate Ethyl Ester Group. Diethyl vinylphosphonate (1 mmol) was dissolved in 1 M aqueous NaOH (20 mL), and the reaction mixture was stirred at rt for 24 h. The reaction mixture was diluted with water (20 mL) and passed through a column of Dowex 50 H + form (20 g). The column was washed with water (20 mL) and ethanol (40 mL). Acidic eluate was concentrated in vacuo and co-evaporated with ethanol (2 × 20 mL).
General Method B1: Esterification of Monomethyl Vinylphosphonate Using Oxalylchloride. Mono alkyl vinylphosphonate (1 mmol) was rendered dry by co-evaporation with EtOH (10 mL/mmol) and toluene (10 mL), dissolved in DCM (3 mL), and cooled to −78°C under an argon atmosphere. Oxalyl chloride (2 M in DCM) (0.3 mL) was slowly added, and the reaction mixture was stirred at rt for 30 min. A catalytic ammount of DMF (50 μL) was added, and the reaction mixture was stirred until gas evolution ceased. Hydroxyderivative (1 mmol) was then added followed by addition of triethylamine (1.1 mmol). The reaction mixture was stirred at rt for 12 h under an argon atmosphere. The reaction mixture was extracted with sat. soln. NaHCO 3 (10 mL) and sat. soln. NaCl (10 mL). The organic phase was dried over Na 2 SO 4 and concentrated in vacuo. The product was obtained by column chromatography using a linear gradient of acetone in toluene or a linear gradient of C1 in chloroform.
General Method B2: Esterification of Monomethyl Vinylphosphonate Using TPSCl. Mono alkyl vinylphosphonate (1 mmol) and hydroxyderivative (2 mmol) were rendered anhydrous by co-   evaporation with DCM (2 × 10 mL) and dissolved in the same solvent (5 mL). Methylimidazole (3 mmol) and TPSCl (2 mmol) were added, and the reaction mixture was stirred at rt under an argon atmosphere for 24−48 h. The progress of the reaction was followed by TLC using a mixture of acetone/toluene (1:1). The reaction mixture was diluted with DCM (10 mL) and washed subsequently with sat. soln. NaHCO 3 (10 mL) and brine (10 mL). Organic phases were combined, dried over Na 2 SO 4 , and concentrated in vacuo. The product was obtained by column chromatography using a linear gradient of acetone in toluene or a linear gradient of C1 in chloroform. General Method C: Reaction of Monoalkyl Vinylphosphonate with α,ω-Dibromoalkane. Mono alkyl vinylphosphonate (1 mmol) and tetrabutylammonium hydroxide (1 mmol) were rendered anhydrous by co-evaporation with ethanol (2 × 10 mL) and DMF (10 mL) and dissolved in DMF (5 mL). α,ω-Dibromoalkane (0.36 mmol) was added, and the reaction mixture was stirred under an argon atmosphere at 90°C for 24−48 h. The progress of the reaction was followed by TLC using a mixture of acetone/toluene (1:1). The reaction mixture was concentrated in vacuo, and the product was obtained by column chromatography using a linear gradient of acetone in toluene.
General Method D: Michael Addition. The mixture of vinylphosphonate dimer (1 mmol) and secondary amine (3 mmol) in nbutanol (50 mL/mmol) was stirred at 105°C for 24−72 h in a sealed flask. The progress of the reaction was followed by TLC using mixture C1. The reaction mixture was concentrated in vacuo, and the product was obtained by column chromatography using a linear gradient of C1 in chloroform.
General Method E: Removal of Boc Protecting Groups. The starting Boc derivative (1 mmol) was dissolved in 0.5 M methanolic HCl (60 mL). The reaction mixture was stirred at rt for 24 h. The reaction mixture was concentrated in vacuo, and the product was obtained by precipitation from anhydrous ethyl acetate. If necessary, the final product is repurified by preparative HPLC on reversed phase using a linear gradient of methanol in 0.1% aqueous TFA followed by several codistillations with 0.5 M methanolic hydrogen chloride.
General Method F: Guanidination. 1H-Pyrazole-1-carboxamidine hydrochloride (3 mmol) was added to the mixture of LPPO (1 mmol) and ethyldiisopropylamine (6 mmol) in DMF (10 mL) and stirred at rt for 24 h. The reaction mixture was concentrated in vacuo and purified by HPLC on reversed phase using a linear gradient of methanol in 0.1% aqueous TFA followed by several codistillations with 0.5 M methanolic hydrogen chloride.

Hexane-1,6-diyl Bis((Z)-oct-3-en-1-yl) Bis((2-(bis(2-aminoethyl)amino)ethyl)phosphonate) Hexahydrochloride
Hydrophobicity Index (CHIg). The gradient chromatography hydrophobicity index (CHIg) was measured by the linear gradient HPLC method and calculated based on retention time and acetonitrile composition, as described before. 31 Majority of samples were measured using gradient A; for more polar samples, gradient B was used. Analytes were identified by mass spectrometry in full scan mode. The mobile phase was adjusted with 0.1% formic acid to help on elution and ionization of analytes; the final pH was 3.7. HPLC method: CHIg was measured on UPLC-qTof (Waters, Milford, USA) using a C18 UPLC column (Waters XBridge 50 × 2.1 mm, 1.9 μm) with the following gradients: Gradient A: 10% B hold for 0.5 min, 95% B in 5.5 min, hold till 6 min; gradient B: 5% B hold for 1 min, 95% B in 5 min, hold till 5.5 min. A = 0.1% formic acid, B = 0.1% formic acid in acetonitrile. Flow rate was set to 0.5 mL, and injection volume was 0.5 μL. Output signal was monitored by mass spectrometry with positive electrospray ionization in full scan mode.
Membrane Potential Measurements. The change in membrane potential by LEGO-LPPOs was monitored with a DiSC 3 (5) fluorescent probe as described previously. 47 The probe was incorporated to polarized membranes, which leads to a reduction of its fluorescence intensity. When the membrane potential is disrupted by the action of the membrane active substance, the probe is released from the membrane and the fluorescence intensity increases. 48 The bacterial cells (E. coli cells CCM 3954 or S. aureus cells CCM 4223) were grown to OD 450 = 0.2 (corresponding to 4−5 × 10 7 cells/mL), centrifuged (5000g, 25°C, 10 min), and washed twice in glucose buffer (10 mM HEPES, 0.5% glucose). EDTA solution was added to the E. coli suspension to a final concentration of 10 mM to disrupt the outer membrane and facilitate access of the staining of the cytoplasmic membrane. The suspension was incubated with EDTA for 20 min on a roller tube mixer, and the suspension was centrifuged to remove EDTA. The supernatant was discarded, and the resulting pellet was resuspended in glucose buffer to a final OD 450 = 0.2. The DiSC 3 (5) probe (1 mM stock solution in DMSO) was added to this suspension to a final concentration of 1 μM, and the aerated suspension was labeled for 90 min in the dark. The preparation of S. aureus cells was similar, omitting the step with EDTA. Fluorescence intensity was measured at 25°C using a FluoroMax-3 spectrofluorometer (Jobin Yvon, Horiba) with 600 nm excitation and 670 nm emission wavelengths. The RPB590-610 and RPE650LP optical filters were used (Omega Optical) in the excitation and emission paths, respectively. The cell suspension was measured in 10 × 10 mm quartz cuvettes in a volume of 2 mL with continuous stirring by a magnetic stirrer. The LEGO-LPPO from the stock solution was added to the cuvette to the desired concentrations. As a positive control for membrane depolarization, 5 μM melittin (Sigma) was added to the cuvette (not shown). Representative kinetics are shown (n = 10).
Membrane Permeabilization Assay. E. coli CCM 3954 cells were grown aerobically in an LB medium at 37°C to mid log phase (OD 450 = 0.5), harvested (8000g, 25°C, 10 min), washed, and resuspended (final OD 450 = 0.1) in a buffer containing 10 mM HEPES (pH 7.2), 0.5% glucose, and 10 μM propidium iodide (PI, Invitrogen). LPPOs were added to 2 mL of the bacterial suspension in a 10 × 10 mm quartz cuvette, and PI uptake into cells (indicating membrane permeabilization) was monitored as the increase in fluorescence intensity (excitation at 515 nm, emission at 620 nm with bandpass 5 and 5 nm, respectively) at 25°C using a FluoroMax-3 spectrofluorometer (Jobin Yvon, Horriba). The optical filters 3RD500-530 and 3RD570LP (Omega Optical) were used in excitation and emission paths for suppression of light scattered by the cells. The bacterial suspension was continuously stirred by the magnetic stirrer during the measurements. As a positive control for cell permeabilization, 5 μM melittin (Sigma) was added to the cuvette. Representative kinetics are shown (n > 5).
Planar Lipid Bilayer Experiments. Black lipid bilayer membranes were formed by painting a solution of 3% w/v 1,2-diphytanoyl-snglycero-3-phospho-(1′-rac-glycerol) (DPhPG, Avanti Polar Lipids) in n-decane/butanol (9:1, v/v) across the aperture (0.5 mm in diameter) in the diaphragm dividing the Teflon chamber into two compartments. Both compartments contained 1.5 mL of 1 M KCl and 10 mM Tris, pH 7.4. The temperature was kept at 25°C. LPPO was added to the cis side of the membrane in the concentration of 1.25, 2.5, or 5.0 mg/L. The membrane current was registered with Ag/AgCl electrodes (Theta) with a membrane voltage of 50 mV, amplified by an LCA-200-100GV amplifier (Femto), and digitized by a KPCI-3108 card (Keithley). The signal was processed with the QuB software. 49 The histograms of membrane currents were created using kernel density estimation (rectangular kernel with a 5 pS width).
Liposome Preparation and Liposome Leakage Assay. Dioleylphosphatidylglycerol (DOPG), dioleoylphosphatidylethanolamine (DOPE), and dioleoylphosphatidylcholine (DOPC) were purchased from Avanti Polar Lipids. Liposomes for the carboxyfluorescein (CF) leakage assay were prepared by mixing the appropriate amount of lipids (0.5 mg/mL) in chloroform/methanol 2:1 (v/v). The solvent was subsequently evaporated in vacuo to form a thin film on the walls of a glass tube. The multilamellar vesicles were prepared by hydration of lipids in a buffer containing 50 mM 5(6)-carboxyfluorescein (CF) and 5 mM HEPES (pH 7.4) for 90 min. Large unilamellar vesicles (LUVs) were prepared by repeated extrusion of the multilamellar vesicles through 100 nm polycarbonate filters (Avestin) using a Mini-Extruder apparatus (Avanti Polar Lipids). Vesicles were separated from the nonencapsulated dye by gel filtration on Sephadex G-50 using 100 mM NaCl, 0.5 mM Na 2 EDTA, and 5 mM HEPES (pH 7.4) as the elution buffer. Fractions with the highest content of entrapped dye were put together and diluted in the same buffer to give a final phospholipid concentration of 10 μM according to the assessed content of inorganic phosphate. The leakage of CF from suspension of liposomes in 2 mL cuvette was initiated by LPPO addition and monitored as the increase in CF fluorescence intensity (excitation at 480 nm, emission at 520 nm with 2 nm bandpasses) at 25°C using a FluoroMax-3 spectrofluorometer (Jobin Yvon, Horriba). The maximum intensity (100%) was achieved by addition of 0.2% Triton X-100 to liposomal suspension. Representative kinetics are shown (n > 8).
Scanning Electron Microscopy. All bacterial samples were essentially processed as described in Sǐkováet al. 50 with some modifications (Pospísǐl et al.). 33 The bacterial suspension in the Mueller−Hinton medium was briefly prefixed in 3% glutaraldehyde for 15 min. Prefixed cells were centrifuged at 5250g for 10 min at room temperature, resuspended in 3% glutaraldehyde in cacodylate buffer (pH 7.2−7.4), and stored in a refrigerator for 24 h. Fixed bacteria were extensively washed in cacodylate buffer and sedimented overnight onto the circular, poly-L-lysine treated glass coverslips. Washed coverslips were post-fixed in 1% OsO 4 in ddH 2 O at room temperature for 1 h, dehydrated in graded ethanol series, and critical point dried in a K850 Critical Point Dryer (Quorum Technologies Ltd., Ringmer, UK). Dried coverslips sputter-coated with 3 nm of platinum (Q150T Turbo-Pumped Sputter Coater; Quorum Technologies Ltd., Ringmer, UK) were examined in an FEI Nova NanoSEM scanning electron microscope (FEI, Brno, Czech Republic) at 3 kV using CBS and TLD detectors.
Determination of Cytotoxicity. Cytotoxicity was assessed by the alamarBlue assay (Invitrogen) with human liver HepG2 cells in a 384well plate format. Cells were incubated with test compound concentrations (0.005−99.0 μM) for 24 h in a supplemented RPMI1640 medium, the medium was removed, and alamarBlue was added followed by incubation for 4 h. Metabolic formation of the fluorescent resorufin was measured on a plate reader (excitation 550 nm, emission 595 nm). The fluorescence signal is proportional to metabolically active and viable cells. Finally, the cytotoxic dose at 50% viability (CTD 50 ) values were calculated reflecting the test compound concentration that reduced cell viability by 50%. Mean values of four replicates are shown. tissue viability cutoff value, i.e., tissue viability ≤60% in the EpiOcular Eye Irritation Test, no prediction can be made from this result in isolation. This is because in case of a true positive, the method cannot resolve between UN GHS Categories 1 and 2. Furthermore, RhCE test methods show a high percentage of false-positive results; therefore, further information will be required for classification purposes according to the IATA guidance document (OECD, 2018. Guidance Document on an Integrated Approach on Testing and Assessment for Serious Eye Damage and Eye irritation. Series on Testing and Assessment No. 263. ENV Publications, Organisation for Economic Cooperation and Development, Paris).